Programmed ribosomal frameshift (PRF) events most commonly induce translating ribosomes to slip by a single base in either the 5′ (−1) or 3′ (+1) direction, though examples of ribosomal “hops”, “shunts”, and “bypasses” have also been documented (reviewed in Jacks, 1990; Farabaugh, 1996; Gesteland & Atkins, 1996). Such translational recoding signals have been valuable in addressing questions relating to ribosome structure and function. For viruses that utilize PRF, the efficiencies of frameshift events are critical: they determine the stoichiometry of viral structural to enzymatic proteins available for virus particle assembly, and altering PRF frequencies have dire consequences for virus propagation (reviewed in Dinman et al., 1998). Thus, it is important to understand how frameshifting efficiencies are controlled. The most widespread mechanisms involve inducing ribosomes to stall with their associated tRNAs positioned over specific mRNA sequences called “slippery sites” such that, in the event of slippage, the tRNAs are able to base pair with the out-of-frame codon or codons. Though the cis-acting signals are relatively well characterized, the trans-acting factors and the biophysical parameters that contribute to determine PRF efficiencies are less well understood. Genetic, biochemical, molecular, and pharmacological methods have been employed toward this end. In general, parameters that can affect PRF efficiencies include: 1) changes in the residence time of ribosomes at a particular PRF signal and the precise steps of the elongation cycle that such kinetic changes might occur; 2) changes in the stabilities of ribosome-bound tRNAs and/or ribosome catalytic function due to alterations in intrinsic ribosomal components such as ribosomal proteins, rRNAs, and codon:antidcodon interactions; and 3) defects in the abilities of the translational apparatus to recognize and correct errors (reviewed in Harger et al., 2002).
The genetic manipulability of the yeast Saccharomyces cerevisiae has facilitated the identification of trans-acting factors that can affect frameshifting efficiencies, and researchers in the field have capitalized on the presence of two endogenous viruses of yeast to this end. The Ty1 retrotransposable element of yeast utilizes a programmed +1 frameshift to synthesize its Gag-pol precursor (Clare et al., 1988; Belcourt & Farabaugh, 1990), and changes in +1 PRF efficiencies have inhibitory effects on Ty1 retrotransposition frequencies (Xu & Boeke, 1990; Kawakami et al., 1993; Balasundaram et al., 1994; Tumer et al., 1998; Harger et al., 2001; Hudak et al., 2001). The 4.6 kb dsRNA L-A virus of yeast utilizes a programmed −1 ribosomal frameshift to produce its Gag-pol fusion protein (Icho & Wickner, 1989; Dinman et al., 1991; Tzeng et al., 1992), and changes in −1 PRF efficiencies promote loss of the killer phenotype as a consequence of loss of the 1.6-1.8 kb dsRNA M1 satellite virus that encodes the secreted killer toxin (reviewed in Wickner, 1996). It has been previously reported that −1 PRF efficiencies were specifically elevated in cells harboring the mak8-1 allele of RPL3 (Peltz et al., 1999), thus providing an explanation for the original observation that mak8-1 cells could not maintain the killer phenotype (Wickner & Leibowitz, 1974; Wickner et al., 1982). Two alleles of RPL3 have been heretofore described: the tcm1-1 allele contains a single missense mutation changing tryptophan at position 255 to cystine (Fried & Warner, 1981), and the mak8-1 allele contains this mutation plus a second missense mutation changing proline at position 257 to threonine. In Example 1 herein, the effects of single and double mutations at this site on −1 PRF, killer virus maintenance, and peptidyltransferase activities in isogenic rpl3 gene deletion strains, are described. A PCR-based mutagenesis approach is employed to identify and characterize a new allele of RPL3 consisting of mutation of isoleucine 282 to threonine (I282T) that was unable to maintain the yeast killer virus. All of the mak8 alleles promoted increased −1 PRF efficiencies, and ribosomes isolated from cells expressing these alleles had decreased peptidyltransferase activities Molecular modeling based on the Haolarcula marismortui 50S ribosomal subunit (Ban et al., 2000) reveals that W255 is the closest amino acid residue in the ribosome to the peptidyltransferase center active site, that P257 is required to form an important bend in a loop that positions W255, and that 1282 is in the hydrophobic core at the base of the loop. How these structural changes might specifically affect peptidyltransferase function and −1 PRF is discussed within the context of the recent explosion of information pertaining to ribosome structure and function. Further, based on a recent study showing that deletion of ribosomal protein L41 results in peptidyltransferase defects, both −1 and +1 PRF in isogenic rpl41-deficient and wild-type strains were assayed as described herein (see, Example 1). The finding that −1 PRF was also specifically stimulated in rpl41-deficient strains provides additional evidence that −1PRF efficiencies can be influenced by peptidyltransfer rates.